T-cells recognize and respond to specific peptide antigens presented on antigen presenting cells (APC). For autoreactive T-cells, antigen-specificity is directed against peptides derived from self-proteins. Upon recognition of self-peptide, autoreactive T-cells can become activated, leading to tissue destruction and autoimmune disease. Alternatively, upon ligand recognition, autoreactive T-cells can become tolerized, a condition marked by unresponsiveness upon future antigenic challenge. It remains unclear as to the parameters that determine whether an autoreactive T-cell will be activated or induced to undergo tolerance. Using an antigen and TCR double LCMV transgenic diabetes model (RIP-gp/P14), we have found that the induction of autoimmune disease versus tolerance is a function of the activation state of the APCs where activated APC promote autoimmunity and resting APC promote tolerance. Surprisingly, however, the induction of autoimmune disease by activated APC was independent of the co-stimulatory molecule, CD28. We believe that T- cell activation was achieved as a result of additional molecular interactions provided by the activated APC, which may amplify the TCR signal in the absence of co-stimulation. We hypothesize, therefore, that T-cell activation can be "tuned" in which the lowering of signal strength through one molecular interaction can be compensated by increasing the signal strength through a number of other molecular interactions. Moreover, we hypothesize that "tuning" of T-cell activation may affect the induction of tolerance versus autoimmunity. We will address our first hypothesis in Aim 1 by assessing T-cell activation, autoimmune disease induction, and tolerance induction upon "tuning" or altering the strength-of-signal through different T-cell-APC interactions. We have also found, utilizing TRAF6-deficient APCs to present LCMV-gp peptide to P14 transgenic T-cells, that when the APC cannot signal through TRAF6, the APC are more susceptible to apoptosis and are poorer inducers of T-cell responses in vitro. We hypothesize that the balance between the induction of tolerance and autoimmunity may also be a function of the lifespan of the APC presenting self-antigen, where prolonged survival promotes the induction of autoimmunity and shortened survival promotes the induction of tolerance. Our second hypothesis will be addressed in Aim 2 by assessing the mechanism(s) for regulating APC survival upon antigen presentation and assessing the induction of autoimmunity and tolerance when utilizing APC of prolonged versus shortened survival ability. The overall goal of this proposal is to further investigate the characteristics of APCs that influence the activation of pathogenic autoreactive T-cells in hopes of understanding the cellular conditions that promote autoimmunity over tolerance and to identify potential targets for the prevention of autoimmune disease.